International Journal of
Molecular Sciences
Review
Cytokinesis Failure Leading to Chromosome
Instability in v-Src-Induced Oncogenesis
Yuji Nakayama 1, *, Shuhei Soeda 2 , Masayoshi Ikeuchi 1 , Keiko Kakae 1 and Naoto Yamaguchi 2
1
2
*
Department of Biochemistry & Molecular Biology, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan;
[email protected] (M.I.); [email protected] (K.K.)
Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University,
Chiba 260-8675, Japan; [email protected] (S.S.); [email protected] (N.Y.)
Correspondence: [email protected]; Tel.: +81-75-595-4653; Fax: +81-75-595-4758
Academic Editors: Vassilis G. Gorgoulis, Athanassios Kotsinas, Alexandros G. Georgakilas, Ioannis Trougakos
and Aristides Eliopoulos
Received: 22 February 2017; Accepted: 9 April 2017; Published: 12 April 2017
Abstract: v-Src, an oncogene found in Rous sarcoma virus, is a constitutively active variant of c-Src.
Activation of Src is observed frequently in colorectal and breast cancers, and is critical in tumor
progression through multiple processes. However, in some experimental conditions, v-Src causes
growth suppression and apoptosis. In this review, we highlight recent progress in our understanding
of cytokinesis failure and the attenuation of the tetraploidy checkpoint in v-Src-expressing cells. v-Src
induces cell cycle changes—such as the accumulation of the 4N cell population—and increases the
number of binucleated cells, which is accompanied by an excess number of centrosomes. Time-lapse
analysis of v-Src-expressing cells showed that cytokinesis failure is caused by cleavage furrow
regression. Microscopic analysis revealed that v-Src induces delocalization of cytokinesis regulators
including Aurora B and Mklp1. Tetraploid cell formation is one of the causes of chromosome
instability; however, tetraploid cells can be eliminated at the tetraploidy checkpoint. Interestingly,
v-Src weakens the tetraploidy checkpoint by inhibiting the nuclear exclusion of the transcription
coactivator YAP, which is downstream of the Hippo pathway and its nuclear exclusion is critical in
the tetraploidy checkpoint. We also discuss the relationship between v-Src-induced chromosome
instability and growth suppression in v-Src-induced oncogenesis.
Keywords: v-Src; cytokinesis; tetraploidy checkpoint; YAP; chromosome instability
1. Introduction
v-Src is an oncogene that was found in Rous sarcoma virus and its cellular counterpart is c-Src [1,2].
In v-Src, a C-terminal tyrosine residue that is phosphorylated by C-terminal Src kinase (Csk) and is
responsible for the closed- and inactive-conformation is lost, resulting in the constitutive activation of
v-Src. Src activation is very common in colorectal and breast cancers and is frequently critical in tumor
progression through multiple processes, including migration, invasion, anoikis resistance, disruption
of cadherin-mediated cell-cell contacts, proliferation, and resistance to apoptosis [1,3]. However,
in some experimental conditions, v-Src causes growth suppression [1,4].
Growth promoting factor-induced growth suppression has been reported. The addition of
epidermal growth factor (EGF) to serum-free and serum-containing cell culture media results in
a marked inhibition of cell proliferation in the A431 human epidermoid carcinoma cell line [5–7].
The growth of estrogen-independent breast cancer MDA-MB-468 cells—which overexpress the EGF
receptor (EGFR)—is inhibited by EGF; this is accompanied by the increased expression of some
genes, including c-myc [8]. Upon treatment with EGF, fibroblastic cells or mammary tumor cells
overexpressing EGFR and ErbB2 undergo apoptosis in a p38-dependent manner [9]. Other pathways
Int. J. Mol. Sci. 2017, 18, 811; doi:10.3390/ijms18040811
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downstream of the EGFR are also involved in the suppression of cell proliferation. Ras provokes
responses to cause cell cycle arrest or apoptosis, although Ras suppresses c-myc-induced apoptosis
through the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway [10,11]. Constitutively
activated Raf-1, which is a proto-oncogene and a downstream kinase of Ras, increases apoptosis in
breast cancer MCF-7 cells [12]. The deregulated expression of c-myc induces apoptosis, although
it confers the ability to proliferate in low serum [13,14]. E2F-1 and v-Jun promote both cell cycle
progression and apoptosis [15,16]. In addition, the adenovirus protein E1A stimulates transformed
focus formation and induces apoptosis, which is inhibited by the E1B protein [17].
Similarly to these growth-promoting factors, v-Src has been reported to cause growth inhibition
despite its oncogenic effects. The induced expression of the constitutively active mutant c-SrcY527F
has a negative effect on the proliferation of human colorectal cancer HCT116 and SW480 cells in vitro
and tumor growth in a xenograft model in vivo [18]. Cell cycle analysis showed the accumulation
of cells in the G2 phase of the cell cycle with increased phosphorylation of Tyr15 in Cdk1 and
decreased phosphorylation of Ser10 in histone H3 [18]. In Rat-1 fibroblast cells, v-Src does not
accelerate the proliferation rate in spite of the decreased expression of the Cdk inhibitor p27 [19].
In carcinoma AA/C1/AB10 cells, an increase of EGFR expression enhances c-Src kinase activity,
resulting in increased motility, but not growth stimulation [20]. Furthermore, when Ras and PI3K are
inhibited simultaneously, v-Src induces apoptosis in a p53-independent manner [21,22].
Growth suppression and cell death following the hyper-induction of growth factor signaling are
thought to be cellular responses to suppress the propagation of unfavorable genes, since activated
Ras induces DNA double strand breaks, and other oncogenes, such as myc and E2F1, have similar
effects [23,24]. If this is the case, it is thought that growth factor signaling requires further alterations of
the gene profiles of primary cells to escape from growth factor-induced growth suppression. One way
of altering gene profiles is via changes in gene expression, which is accomplished by gene usage at the
transcriptional level, which is observed in v-Src-expressing cells [25]. Another way to alter gene profiles
is genetic diversification through chromosome instability. Chromosome instability can be caused by the
aberrant segregation of chromosomes during cell division. However, only a few reports have examined
the effect of v-Src on genetic stability. In this review we describe our recent findings, including the
effect of v-Src on cell proliferation, cytokinesis, and attenuation of the tetraploidy checkpoint, and then
discuss the relationship between v-Src-induced chromosome instability and growth suppression in
v-Src-induced oncogenesis.
2. v-Src Suppresses Cell Proliferation and Induces Tetraploidization
We generated three cell lines derived from mouse fibroblast NIH3T3, human cervix HeLa S3,
and human colorectal HCT116 cells—which can induce the expression of v-Src upon treatment with
the tetracycline analog doxycycline (Dox)—to observe the effect of v-Src on cell proliferation [4].
When these cells were treated with Dox, v-Src was expressed; phosphorylation of the tyrosine residues
in a large number of proteins was enhanced. Cell–cell attachments were weakened, which was
accompanied by the loss of E-cadherin staining from the plasma membrane, and the cells were
round-shaped. These are the typical features of v-Src expression. v-Src caused growth suppression
in these cells, and flow cytometry analysis revealed the alteration of cell cycle progression. While a
low concentration of Dox caused the slight accumulation of cells with more than 4N DNA, a high
concentration of Dox caused the transient accumulation of 4N cells and then an increase in the sub-G1
population, indicating the induction of apoptosis.
It is noteworthy to mention that the expression level of v-Src in these cells depends on the Dox
concentration and the duration of induction. When these cells are treated with a high concentration of
Dox, despite induction for only a short period, v-Src is expressed at high levels and induces a variety
of effects, including the detachment of the cells from the culture dish. This makes it difficult to perform
observations under a microscope. Furthermore, it induces growth suppression and apoptosis as
described above. In contrast, a low concentration of Dox induces moderate increases in protein-tyrosine
Int. J. Mol. Sci. 2017, 18, 811
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phosphorylation and causes some phenotypes; however, it causes neither detachment of cells nor
growth suppression. Thus, these cell lines are thought to be very useful for studying the effect of v-Src
at different expression levels without detachment. Furthermore, it is thought that each phenotype
caused by v-Src has its own threshold of tyrosine phosphorylation. We chose the concentration of Dox
and duration of induction that resulted in less of an effect on both cell proliferation and attachment to
the culture dish, but also made it possible to observe the cells under a microscope.
In order to elucidate how 4N cells accumulate following v-Src expression, the cells were treated
with the low concentration of Dox for 6 days. Microscopic analysis showed the accumulation of
binucleated cells, suggesting that the accumulation of 4N cells is not attributed to G2-arrest. Taken
together with the increase in the number of centrosomes, these binucleated cells are generated through
cytokinesis failure. Time-lapse analysis of v-Src-expressing cells confirmed that cytokinesis failure is
caused by the regression of the cleavage furrow, resulting in the accumulation of 4N cells.
Src family kinases are known to regulate cell division. Upon mitotic entry, Cdk1 induces
phosphorylation of the unique domain of Src family kinases, leading to up-regulation of their kinase
activities [26–30]. Mitotic entry is inhibited by microinjection of anti-Src antibody into G2 cells [31],
and inhibition of Src family kinases blocks mitotic progression in prophase [32]. Cytokinesis is also
inhibited by microinjection of anti-Src antibody or Src homology 2 (SH2) domain of Src, or by treatment
with the Src inhibitor PP2 [33–35]. These results suggest that Src family kinases are required for
proper mitotic progression. Each member of the Src family kinases appears to have a distinct role
in cell division. Fyn, a member of Src family kinases, participates in the assembly of mitotic spindle
microtubules and accelerates mitotic progression [36]. c-Src promotes proper spindle orientation
in early prometaphase [37]. Because kinase activities are tightly regulated in a spatiotemporal
manner during cell division, we had expected that v-Src—which is aberrantly and constitutively
activated—gives rise to uncontrolled cell division including cytokinesis failure.
3. v-Src-Induced Cytokinesis Failure Is Caused through Delocalization of Mitotic Regulators
Cytokinesis is the final process that divides cell contents into 2 cells (see reviews, [38,39]). During
this process, a signal is transferred from the central spindle to the equatorial cortex. Central spindle
formation is regulated by the chromosomal passenger complex (CPC), which consists of Aurora B,
INCENP, Survivin, and borealin, but also requires PRC1, KIF4, and Mklp1. Mklp1, a component of
the centralspindlin complex, is phosphorylated at Ser708 by Aurora B and is thereby recruited to the
spindle midzone. The CPC and centralspindlin complex play roles in contractile ring assembly at
the equatorial cortex. After the constriction of the contractile ring, the cell is finally separated into
two daughter cells through a final step called abscission, in which ESCRT-III proteins catalyze the
scission of membrane necks. Abscission timing is regulated by Plk1 and Aurora B kinases, and Aurora
B prevents premature abscission if chromosome bridges are present in the intracellular bridges.
In order to explore the mechanism underlying v-Src-induced cytokinesis failure, we examined the
localization of cytokinesis regulators and found the delocalization of the kinesin motor proteins
Mklp1 and Mklp2, and the components of the CPC—Aurora B kinase and INCENP—from the
anaphase midzone (Figure 1) [4]. Cleavage furrow ingression requires the localization of Mklp1;
v-Src-induced cytokinesis failure is caused by Mklp1 delocalization. Given that v-Src downregulates
cell adhesion [4,40], and that cell adhesion interacts with cytokinesis [41,42], v-Src-induced cytokinesis
failure would be caused by detachment of v-Src-expressing cells. We examined suspension cultures of
HeLa S3 cells to exclude the possibility that the detachment of v-Src-expressing cells is involved in
v-Src-induced cytokinesis failure. Similar to cells attached to culture dishes, Aurora B was localized to
the anaphase midzone in the absence of v-Src expression and delocalized upon v-Src expression in
suspension-cultured HeLa S3 cells, excluding the possibility that cytokinesis failure is caused by the
loss of adhesion of v-Src-expressing cells.
How are cytokinesis regulators delocalized by v-Src? A plausible hypothesis is the inhibition
of the kinesin-like motor protein Mklp2, which is responsible for the relocation of the CPC from
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the centromeres to the anaphase spindle midzone [43]. Knockdown of Mklp2 causes failure of
abscission, generating
[44].
In the
absence
of Mklp2,
Mklp1Mklp1
phosphorylation
at Ser708,
generatingbinucleated
binucleatedcells
cells
[44].
In the
absence
of Mklp2,
phosphorylation
at
which iswhich
required
the recruitment
Mklp1 to the
lost midzone,
[45]. This phosphorylation
is
Ser708,
is for
required
for the of
recruitment
of midzone,
Mklp1 tois the
is lost [45]. This
catalyzed by Aurora
B, and when
Mklp2 isB,knocked
down,
Aurora
B cannotdown,
relocate
to the anaphase
phosphorylation
is catalyzed
by Aurora
and when
Mklp2
is knocked
Aurora
B cannot
spindle to
midzone
and remains
segregating
chromosomes
at anaphase
[43]. Thus,
Aurora B
relocate
the anaphase
spindlewith
midzone
and remains
with segregating
chromosomes
at anaphase
cannot
phosphorylate
Mklp1phosphorylate
at Ser708, resulting
delocalization
of the
Mklp1
and therebyofcausing
[43].
Thus,
Aurora B cannot
Mklp1inatthe
Ser708,
resulting in
delocalization
Mklp1
cytokinesis
this
hypothesis
is unlikely
to be true,issince
Aurora
neversince
co-localizes
and
therebyfailure.
causingHowever,
cytokinesis
failure.
However,
this hypothesis
unlikely
to beB true,
Aurora
with
segregating
chromosomes
upon v-Src
expression.
B
never
co-localizes
with segregating
chromosomes
upon v-Src expression.
Figure
1. v-Src
Figure 1.
v-Src induces
induces cytokinesis
cytokinesis failure.
failure. The
The chromosomal
chromosomal passenger
passenger complex
complex (CPC)
(CPC) and
and
centralspindlin
complex
are
localized
at
the
spindle
midzone;
however,
v-Src
causes
delocalization
centralspindlin complex are localized at the spindle midzone; however, v-Src causes delocalization
of
of the
the components
components of
of these
these complexes.
complexes. As
As aa result,
result, cytokinesis
cytokinesis fails
fails due
due to
to regression
regression of
of the
the cleavage
cleavage
furrow,
resulting
in
the
formation
of
binucleated
cell.
furrow, resulting in the formation of binucleated cell.
Interestingly, the delocalization of Aurora B was observed after a 15-min incubation of
Interestingly, the delocalization of Aurora B was observed after a 15-min incubation of
v-Src-expressing cells with the Aurora B inhibitor ZM447439 [4]. This means that the kinase activity
v-Src-expressing cells with the Aurora B inhibitor ZM447439 [4]. This means that the kinase
of Aurora B is required to maintain its localization to the anaphase spindle midzone, in agreement
activity of Aurora B is required to maintain its localization to the anaphase spindle midzone,
with a previous report [46]. When the cell cycle was synchronized by treatment with nocodazole, the
in agreement with a previous report [46]. When the cell cycle was synchronized by treatment with
auto-phosphorylation of Aurora B was not different between v-Src-expressing and
nocodazole, the auto-phosphorylation of Aurora B was not different between v-Src-expressing and
v-Src-non-expressing cells. However, kinase activity should be evaluated in anaphase cells and not
v-Src-non-expressing cells. However, kinase activity should be evaluated in anaphase cells and not
in nocodazole-synchronized cells. Considering that the duration of anaphase is generally short,
in nocodazole-synchronized cells. Considering that the duration of anaphase is generally short,
it seems to be difficult to examine protein modifications at anaphase by western blotting because of
it seems to be difficult to examine protein modifications at anaphase by western blotting because of the
the difficulty of synchronizing the cell cycle at anaphase. However, we developed a method to
difficulty of synchronizing the cell cycle at anaphase. However, we developed a method to synchronize
synchronize cells at anaphase by using a low concentration of nocodazole and blebbistatin and
cells at anaphase by using a low concentration of nocodazole and blebbistatin and optimizing the
optimizing the period of treatment with the reagents, by which approximately 40% of cells were
period of treatment with the reagents, by which approximately 40% of cells were synchronized at
synchronized at anaphase [47]; nevertheless, it is still difficult to synchronize the cell cycle in
anaphase [47]; nevertheless, it is still difficult to synchronize the cell cycle in v-Src-expressing cells due
v-Src-expressing cells due to the changes in the cell cycle [4,18] and partial disruption of the spindle
to the changes in the cell cycle [4,18] and partial disruption of the spindle assembly checkpoint [4].
assembly checkpoint [4]. Thus, after optimization of the protocol for synchronizing v-Src-expressing
Thus, after optimization of the protocol for synchronizing v-Src-expressing cells, further studies
cells, further studies including a search for v-Src substrates would help to understand the
including a search for v-Src substrates would help to understand the mechanism underlying the
mechanism underlying the v-Src-induced delocalization of cytokinesis regulators.
v-Src-induced delocalization of cytokinesis regulators.
4. v-Src Attenuates the Tetraploidy Checkpoint
Although tetraploidy is one of the causes of chromosome instability through the increase in the
number of centrosomes [48,49] and promotes tumorigenesis in p53-null cells [50], tetraploid cells can
be removed by a mechanism called the tetraploidy checkpoint [51–53]. However, we observed a
Int. J. Mol. Sci. 2017, 18, 811
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4. v-Src Attenuates the Tetraploidy Checkpoint
Although tetraploidy is one of the causes of chromosome instability through the increase in the
number of centrosomes [48,49] and promotes tumorigenesis in p53-null cells [50], tetraploid cells
Int. J. Mol. Sci. 2017, 18, 811
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can be removed by a mechanism called the tetraploidy checkpoint [51–53]. However, we observed
a time-dependent
increase
number
binucleated
cells
upon
induced
expression
of v-Src
time-dependent
increase
in in
thethe
number
of of
binucleated
cells
upon
thethe
induced
expression
of v-Src
in
in
human
colon
carcinoma
HCT116
and
mouse
fibroblast
NIH3T3
cells,
which
is
accompanied
by
human colon carcinoma HCT116 and mouse fibroblast NIH3T3 cells, which is accompanied by the
the accumulation
thecell
4Npopulation
cell population
oncytometry
flow cytometry
Furthermore,
we also
accumulation
of theof4N
on flow
analysis.analysis.
Furthermore,
we also observed
observed
an
increase
in
the
number
of
cells
having
multipolar
spindles
with
excess
centrosomes.
an increase in the number of cells having multipolar spindles with excess centrosomes. These results
These results
suggest
that v-Src
causes chromosome
instability
through cytokinesis
and the
suggest
that v-Src
causes
chromosome
instability through
cytokinesis
failure andfailure
the resulting
resulting tetraploidization.
given
that cell
cycle progression
is prevented
in tetraploid
tetraploidization.
However,However,
given that
cell cycle
progression
is prevented
in tetraploid
cells bycells
the
by
the
tetraploidy
checkpoint,
tetraploid
cells
generated
by
v-Src
expression
should
be
arrested
at the
tetraploidy checkpoint, tetraploid cells generated by v-Src expression should be arrested at the
G1
G1 phase
with
DNA
content.
Thus,
increaseininthe
thenumber
numberofofcells
cells with
with multipolar
multipolar spindles
phase
with
4N4N
DNA
content.
Thus,
thethe
increase
spindles
raised the
the possibility
possibility that
raised
that the
the tetraploidy
tetraploidy checkpoint
checkpoint is
is attenuated
attenuated in
in v-Src-expressing
v-Src-expressing cells.
cells.
The
Hippo
pathway
is
reportedly
activated
in
tetraploid
cells;
LATS kinases
kinases are
are activated,
activated,
The Hippo pathway is reportedly activated in tetraploid cells; LATS
resulting in
in the
theinhibition
inhibitionofofthe
thetranscriptional
transcriptional
regulators
YAP
TAZ
stabilization
of
resulting
regulators
YAP
andand
TAZ
andand
the the
stabilization
of p53
p53
(Figure
2)
[53].
In
this
case,
YAP
is
phosphorylated
by
LATS2
and
excluded
from
the
nucleus.
(Figure 2) [53]. In this case, YAP is phosphorylated by LATS2 and excluded from the nucleus. In
In addition,
LATS2
binds
andinhibits
inhibitsMDM2—an
MDM2—anE3
E3ubiquitin
ubiquitinligase
ligase for
for p53—leading
p53—leading to
to p53
p53
addition,
LATS2
binds
to to
and
stabilization [54].
[54]. We
Weobserved
observedthat
thatthe
thesubcellular
subcellular
localization
YAP
NIH3T3
cells
depended
stabilization
localization
of of
YAP
in in
NIH3T3
cells
depended
on
on
cell
density,
suggesting
that
its
subcellular
localization
is
downstream
of
the
Hippo
pathway
in
cell density, suggesting that its subcellular localization is downstream of the Hippo pathway in
NIH3T3 cells
localization
waswas
found
to betopartially
regulated
by Src kinase
NIH3T3
cells[4].
[4].This
Thissubcellular
subcellular
localization
found
be partially
regulated
by Src activity,
kinase
since
Src
inhibition
by
the
Src
inhibitor
PP2
led
to
the
cytoplasmic
localization
of
YAP,
and
activity, since Src inhibition by the Src inhibitor PP2 led to the cytoplasmic localization of YAP,v-Src
and
expression
led to the
of YAP. When
NIH3T3
cellsNIH3T3
were treated
B
v-Src
expression
lednuclear
to the localization
nuclear localization
of YAP.
When
cells with
werecytochalasin
treated with
or the Plk1 inhibitor
the number
of binucleated
cells
increased
through
cytokinesisthrough
failure.
cytochalasin
B or theBI2536,
Plk1 inhibitor
BI2536,
the number
of was
binucleated
cells
was increased
In
these
binucleated
cells,
the
cytoplasmic
localization
of
YAP
was
promoted,
suggesting
activation
cytokinesis failure. In these binucleated cells, the cytoplasmic localization of YAP was promoted,
of the Hippo
pathway. ofHowever,
v-Src
expression
led to the
nuclear
localization
most
suggesting
activation
the Hippo
pathway.
However,
v-Src
expression
led of
to YAP
the in
nuclear
of
the
binucleated
NIH3T3
cells.
In
agreement
with
these
subcellular
localizations,
v-Src
inhibited
localization of YAP in most of the binucleated NIH3T3 cells. In agreement with these subcellular
the phosphorylation
of LATSthe
kinases
and YAP, indicating
v-Srcand
mayYAP,
inhibit
LATS kinases
and
localizations,
v-Src inhibited
phosphorylation
of LATSthat
kinases
indicating
that v-Src
thereby
inhibits
YAP
phosphorylation,
althoughYAP
the v-Src
substrate responsible
for this
may
inhibit
LATS
kinases
and thereby inhibits
phosphorylation,
although the
v-Srcphenotype
substrate
has
not
been
determined.
Therefore,
v-Src
may
weaken
the
tetraploidy
checkpoint
by
attenuating
the
responsible for this phenotype has not been determined. Therefore, v-Src may weaken the
Hippo
pathway.
tetraploidy checkpoint by attenuating the Hippo pathway.
Figure
2. v-Src
v-Src weakens
weakens the
the tetraploidy
tetraploidy checkpoint.
tetraploid
Figure 2.
checkpoint. LATS
LATS kinases,
kinases, which
which are
are activated
activated in
in tetraploid
cells,
phosphorylate
YAP,
leading
to
exclusion
of
YAP
from
the
nucleus.
In
addition,
LATS
activation
cells, phosphorylate YAP, leading to exclusion of YAP from the nucleus. In addition, LATS activation
results
in stabilization
stabilization of
of p53
p53through
throughinhibition
inhibitionofofMDM2.
MDM2.As
Asa aresult,
result,tetraploid
tetraploid
cells
removed
results in
cells
areare
removed
at
at
this
tetraploidy
checkpoint.
v-Src
inhibits
YAP
phosphorylation
by
inhibiting
LATS,
possibly
this tetraploidy checkpoint. v-Src inhibits YAP phosphorylation by inhibiting LATS, possibly through
through
activation
of the
PI3K pathway.
activation
of the PI3K
pathway.
One possible mechanism underlying the v-Src-induced suppression of the Hippo pathway
One possible mechanism underlying the v-Src-induced suppression of the Hippo pathway could
could be phosphorylation of YAP by v-Src. YAP was isolated initially as a Yes-associated protein,
be phosphorylation of YAP by v-Src. YAP was isolated initially as a Yes-associated protein, where YAP
where YAP binds to the Src homology 3 (SH3) domain of Yes, a member of the Src-family kinases
binds to the Src homology 3 (SH3) domain of Yes, a member of the Src-family kinases [55], in a manner
[55], in a manner that depends on Yes kinase activity [56]. YAP is phosphorylated at Tyr357 by Yes
[57], resulting in the nuclear localization of YAP [58]. Abl tyrosine kinase also phosphorylates YAP
at this site, leading to the stabilization of YAP [59]. However, we never observed an increase in the
expression level of YAP upon v-Src expression. Thus, direct phosphorylation of YAP at Tyr357 by
v-Src may not be responsible for the v-Src-induced nuclear localization of YAP in our experimental
Int. J. Mol. Sci. 2017, 18, 811
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that depends on Yes kinase activity [56]. YAP is phosphorylated at Tyr357 by Yes [57], resulting in the
nuclear localization of YAP [58]. Abl tyrosine kinase also phosphorylates YAP at this site, leading to
the stabilization of YAP [59]. However, we never observed an increase in the expression level of YAP
upon v-Src expression. Thus, direct phosphorylation of YAP at Tyr357 by v-Src may not be responsible
for the v-Src-induced nuclear localization of YAP in our experimental conditions. Another possibility
is activation of PI3K by v-Src. The FAK-Src-PI3K pathway reportedly inhibits the Hippo pathway,
leading to a decrease in YAP phosphorylation at Ser127 [60]. Since v-Src is also known as an activator
of PI3K, v-Src may activate PI3K and reduce the activity of LATS kinases.
The tumor suppressor p53 also plays an important role in the suppression of the cell cycle in
tetraploid cells (Figure 2) [54]. We also observed a decrease in p53 levels in v-Src-expressing cells [40].
p53 degradation is regulated by the E3 ubiquitin ligase MDM2 [61–64], which is inactivated by
LATS2 [54]. Because we observed the v-Src-induced reduction of YAP phosphorylation at Ser127,
a substrate of LATS kinases, the inactivation of LATS kinases could be the underlying mechanism;
namely, inactivated LATS kinases cannot prevent MDM2-mediated p53 degradation, leading to
a decrease in p53 levels. Src also reduces p53 levels in a different way; Src inactivates the phosphatase
PP2A by phosphorylating Tyr307, leading to MDM2-mediated p53 degradation through an increase in
MDM2 phosphorylation at Ser166, which enhances its interaction with p53 [65]. Furthermore, strong
growth signals can overcome cell cycle arrest in tetraploid cells [53]. v-Src stimulates growth signals,
such as ERK and Akt, contributing to the silencing of the tetraploidy checkpoint. Taken together,
v-Src may weaken the tetraploidy checkpoint through multiple pathways.
5. v-Src Can Induce Chromosome Instability, Generating Genetic Diversity
One of the causes of chromosome instability is the formation of multipolar spindles [66]. An excess
number of centrosomes lead to the asymmetrical segregation of chromosomes through multipolar
spindle formation and the following multipolar anaphase. However, an excess number of centrosomes
tend to form bipolar spindles by centrosome clustering; that is, centrosomes gather into two poles
after the formation of multipolar spindle intermediates [48]. These plausible bipolar spindles function
incorrectly, since lagging chromosomes are observed frequently during anaphase [48]. For bipolar
spindles with an excess number of centrosomes, merotelic kinetochore-microtubule attachment errors
occur easily, resulting in lagging chromosomes [48,66–68]. Thus, dysregulation of the number of
centrosomes results in chromosome instability.
v-Src induces tetraploid cells through cytokinesis failure. Tetraploid cells have an excess number
of centrosomes, which can form multipolar spindles. Even though pole clustering prevents cells
from forming multipolar spindles, transient multipolar spindle intermediates favor the formation
of merotelic attachments [48]. This can lead to the premature onset of anaphase with lagging
chromosomes, resulting in the formation of a micronucleus [69]. Chromosomes in micronuclei
undergo complex and localized genomic rearrangements through a process called chromothripsis,
in which chromosomes are fragmented and re-ligated by non-homologous end joining [69–71]. Thus,
v-Src-induced cytokinesis failure is a trigger for v-Src-induced genetic diversification.
We reported that non-membrane-bound Src-family kinases induce formation of chromosome
bridges [72], and that v-Src also induces them in a caffeine-dependent manner through induction
of DNA damage [73]. Chromosome bridges result in the generation of daughter cells with different
numbers of chromosomes and thus generate genetic diversity. Furthermore, cytokinesis in cells with
chromosome bridges sometimes fails if the chromosome bridges are not resolved before abscission.
Chromosome bridges activate the abscission checkpoint that prevents cells in the abscission phase
from furrow regression [74]. However, given that the abscission checkpoint requires Aurora B kinase
activity [75–77], v-Src may disrupt the abscission checkpoint and induce furrow regression of cells
with chromosome bridges through delocalization of Aurora B from the midbody. Thus, v-Src can
cause cytokinesis failure more frequently in chromosome bridge-containing cells than in cells without
chromosome bridges.
Int. J. Mol. Sci. 2017, 18, 811
7 of 13
Aurora B regulates the appropriate binding of microtubules and kinetochores; monotelic and
syntelic attachments are corrected at the spindle assembly checkpoint, which requires Aurora B kinase
activity [78,79]. In addition, merotelic attachments are also corrected by a mechanism dependent on
Aurora B and MCAK [80,81]. If the delocalization of Aurora B that was observed in v-Src-expressing
anaphase cells [4] is caused before anaphase onset, it may give rise to erroneous chromosome
segregation through inappropriate microtubule-kinetochore attachments. This can lead to further
Int.
J. Mol. Sci. 2017,
18, 811
7 of 12
cytokinesis
failure.
6. Conclusions and Perspectives
For example,
example,
Genetic alteration through chromosome instability causes growth suppression. For
aneuploid yeast cells show proliferative
proliferative disadvantages,
disadvantages, such
such as
as defects
defects in
in cell
cell cycle
cycle progression
progression [82].
[82].
Mis-segregation of
of chromosomes
chromosomesininhuman
humandiploid
diploid
cells
delays
cycle
progression,
the
Mis-segregation
cells
delays
cellcell
cycle
progression,
and and
the p53
p53 pathway
an important
limiting
proliferationofofaneuploid
aneuploidhuman
humancells
cells [83].
[83].
pathway
playsplays
an important
rolerole
in in
limiting
thethe
proliferation
Although the overexpression of Mad2, which is an essential component of the spindle assembly
G12D delays
checkpoint, together
together with Kras
delays Kras-driven
Kras-driven tumor
tumor initiation,
initiation, Mad2 overexpression
checkpoint,
KrasG12D
facilitates oncogene-independent
oncogene-independent outgrowth
outgrowth and
and tumorigenesis
tumorigenesis in
in mice through chromosome
facilitates
instability [84–86].
[84–86]. This
Thissuggests
suggeststhat
thatchromosome
chromosomeinstability-driven
instability-driven
genetic
diversity
result
instability
genetic
diversity
cancan
result
in
in subclones
and
promote
adaption
a strong
selective
pressure,
such
as the
of growth
signals
subclones
and
promote
adaption
forfor
a strong
selective
pressure,
such
as the
lossloss
of growth
signals
by
by the
withdrawal
oncogene
and
cell
cyclearrest
arrestatatcheckpoints
checkpoints[87–89].
[87–89].Although
Althoughitithas
has long
long been
the
withdrawal
of of
oncogene
and
cell
cycle
malignant progression
progression [1], it also causes
known that v-Src has oncogenic potential and accelerates malignant
suppression, as we observed
observed in some cell lines [4]. We expect that the generation of genetic
growth suppression,
diversity isisrequired
for-v-Src-expressing
cells in
order
overcome
v-Src-induced
growth suppression.
required
for-v-Src-expressing
cells
in toorder
to overcome
v-Src-induced
growth
Here, we propose
model
for v-Src-induced
oncogenesis in
addition toin
the
canonical
roles
of v-Src
suppression.
Here,awe
propose
a model for v-Src-induced
oncogenesis
addition
to the
canonical
(Figure
During
v-Src-induced
oncogenesis,oncogenesis,
v-Src induces
tetraploidization
through cytokinesis
roles
of 3).
v-Src
(Figure
3). During v-Src-induced
v-Src
induces tetraploidization
through
failure. Then,
the activation
the tetraploidy
checkpointcheckpoint
in tetraploid
cells is suppressed
by v-Src,
cytokinesis
failure.
Then, the of
activation
of the tetraploidy
in tetraploid
cells is suppressed
leading
genetictodiversification
through through
chromosome
instability.
Among Among
cells with
broad
by
v-Src,toleading
genetic diversification
chromosome
instability.
cells
withgenetic
broad
diversity,
clones
that
adapt
to
growth-suppressive
circumstances
evolve
and
continue
to
proliferate
or
genetic diversity, clones that adapt to growth-suppressive circumstances evolve and continue to
acquire theor
capacity
metastasize.
proliferate
acquiretothe
capacity to metastasize.
Figure 3. Genetic diversification in v-Src-expressing cells. v-Src induces tetraploidization through
cytokinesis
cytokinesis failure.
failure. The
The activation
activation of
of the
the tetraploidy
tetraploidy checkpoint
checkpoint in
in tetraploid
tetraploid cells
cells is
is suppressed
suppressed by
v-Src. Tetraploid
Tetraploid cells
cells give
give rise
rise to chromosome
chromosome mis-segregation,
mis-segregation, leading
leading to
to genetic
genetic diversification.
diversification.
v-Src causes growth
growth suppression;
suppression;however,
however,among
amongcells
cellswith
withbroad
broadgenetic
genetic
diversity,
cells
resistant
diversity,
cells
resistant
to
to
v-Src-induced
growth
suppression
evolve
continue
to proliferate.
v-Src-induced
growth
suppression
cancan
evolve
andand
continue
to proliferate.
Indeed,
increases in
in Src
Srcactivity
activityare
arefrequently
frequentlyobserved
observedinincolon
colon
and
breast
cancers.
addition
Indeed, increases
and
breast
cancers.
InIn
addition
to
to
the
roles
of
Src
activity
in
tumor
development
and
malignancy
through
a
variety
of
processes,
we
the roles of Src activity in tumor development and malignancy through a variety of processes, we have
have
shown
that
an increase
Src activity
haspotential
the potential
to affect
genetic
stability.
We observed
shown
that an
increase
in Srcinactivity
has the
to affect
genetic
stability.
We observed
that
that
overexpression
of
c-Src
induces
only
slight
increases
in
the
number
of
binucleated
cells
[4],
overexpression of c-Src induces only slight increases in the number of binucleated cells [4], suggesting
suggesting that further activation may be required for induction of cytokinesis failure. As our work
was performed by using v-Src as a model for a constitutively active version of Src, further study is
needed to determine the involvement of c-Src that is activated by a mutation in the C-terminal
inhibitory tyrosine residue or by suppression of Csk activity. Csk is recruited to the vicinity of active
Src in plasma membrane lipid rafts by binding to the transmembrane adaptor protein PAG/Cbp
[90,91]. Thus, PAG/Cbp act as a negative regulator of Src. Src stimulates the PI3K and ERK
Int. J. Mol. Sci. 2017, 18, 811
8 of 13
that further activation may be required for induction of cytokinesis failure. As our work was performed
by using v-Src as a model for a constitutively active version of Src, further study is needed to determine
the involvement of c-Src that is activated by a mutation in the C-terminal inhibitory tyrosine residue
or by suppression of Csk activity. Csk is recruited to the vicinity of active Src in plasma membrane
lipid rafts by binding to the transmembrane adaptor protein PAG/Cbp [90,91]. Thus, PAG/Cbp
act as a negative regulator of Src. Src stimulates the PI3K and ERK pathways, leading to histone
modifications that suppress PAG/Cbp synthesis at the transcriptional level, indicating the presence
of a positive-feedback loop in oncogenic signaling [92]. It would be interesting to examine whether
knockdown of PAG/Cbp expression causes chromosome instability.
v-Src has the potential to induce epithelial-mesenchymal transition (EMT), which contributes
to cancer progression [93–95]. Transcription factors including Snail, zinc-finger E-box-binding (ZEB)
and basic helix-loop-helix (bHLH) drive EMT through repression of the epithelial marker genes and
activation of the mesenchymal phenotype-associated genes [95]. The MEK/ERK pathway participates
in EMT by increasing the expression of EMT transcription factors, and is also linked with the
downregulation of E-cadherin in a manner independent of EMT transcription factors; downregulation
of E-cadherin is a hallmark of EMT, which induces disassembly of adherens junctions. The MEK/ERK
pathway causes overexpression of Cdc6, leading to repression of E-cadherin transcription [96].
Adherens junction maintenance requires the Rac activator Tiam1. During Src-induced disassembly
of adherens junctions, Tiam1 is phosphorylated and degraded through activation of the MEK/ERK
pathway [97]. As previously mentioned, v-Src stimulates the MEK/ERK pathway, suggesting that the
MEK/ERK pathway is involved in the v-Src-induced EMT in both EMT transcription factor-dependent
and -independent manners. As the effects of v-Src-induced genetic diversity on EMT have not yet
been explored, further study is required to determine how chromosome instability caused by v-Src
relates to cancer malignancy, including metastasis, in a mouse model.
Acknowledgments: This work was supported, in part, by Grants-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (16K08253, 25460076) and a grant from the Promotion and Mutual Aid
Corporation for Private Schools of Japan (Kyoto Pharmaceutical University and Chiba University).
Author Contributions: Yuji Nakayama and Naoto Yamaguchi wrote the paper. Yuji Nakayama, Shuhei Soeda,
Masayoshi Ikeuchi, Keiko Kakae and Naoto Yamaguchi discussed and reviewed the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
Csk
EGF
CPC
SH2
SH3
C-terminal Src kinase
epidermal growth factor
chromosomal passenger complex
Src homology 2
Src homology 3
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